Calcium signaling is a critical component of normal immune cell function and development of the heart and brain. The budding yeast Saccharomyces cerevisiae utilizes a calcium signaling mechanism that is closely related to those operating in human cells such as T lymphocytes in their response to antigens. In both of these systems, calcium signals can be sensed by calcineurin, a protein phosphatase that is activated by Ca2+/calmodulin and inhibited by the immunosuppressive drugs Cyclosporin A and FK506. In turn, activated calcineurin dephosphorylates specific transcription factors which then concentrate in the nucleus and induce expression of specific target genes. A new family of proteins conserved from yeast to humans that binds and regulates calcineurin function has been recently identified. One major goal of this proposal is to determine how the yeast protein Rcn1p exerts both positive and negative effects on calcineurin functions. Recent studies also show that yeast maintains a regulatory mechanism related to capacitative calcium entry (CCE) in lymphocytes that helps generate calcium signals. Depletion of Ca2+ from secretory organelles in yeast through inactivation of the Pmr1p Ca2+ pump strongly enhances the activity of a high-affinity Ca2+ channel involving Cch1p, a homolog of voltage-gated Ca2+ channels in animals. Additional goals of this proposal include the identification and characterization of factors involved in releasing Ca2+ from intracellular organelles, sensing Ca2+ concentration in the lumen of these compartments, regulating the Cch1p-dependent Ca2+ channel, and activating another unidentified Ca2+ influx channel in the plasma membrane. Calcineurin- dependent feedback regulation of these Ca2+ channels and of the Ca2+ transporters that dissipate calcium signals also will be evaluated. These goals will be accomplished by combining genetic, molecular, physiological, and biochemical methods. The broad long-term objectives of this project are to elucidate the components of the calcium signaling mechanism operating in all cells and to develop a detailed working model for their interactions and functions.